Cs 56

The distal colon was sampled and fixed in 4% formalin, embedded in paraffin and stained with hematoxylin and eosin (HE) or sirius red as per previous methods [18 (link),24 (link),25 ]. Immunohistochemical staining was performed as reported earlier [25 ]. The following anti-mouse Abs were used: F4/80 (Caltag), ER-TR7 (BMA), Ki-67 (COSMO-Bio), CS56 (Seikagaku) and α-SMA (Abcam). Anti-mouse monoclonal Ab to CHST15 was made in house. Histological score was measured by summation of the following parameters [18 (link)]. Epithelium score was as follows; 0 = normal morphology; 1 = loss of goblet cells; 2 = loss of goblet cells in large areas; 3 = loss of crypts; and 4 = loss of crypts in large areas. Infiltrate score was as follows; 0 = no infiltrate; 1 = infiltrate around crypt basis; 2 = infiltrate reaching the L. muscularis mucosae; 3 = extensive infiltration reaching the L. muscularis mucosae and thickening of the mucosa with abundant edema; and 4 = infiltration of the L. submucosa. For quantification of immunostained and sirius-red stained areas, bright field images were captured using a digital camera (DFC280, Leica Microsystems) at 200-fold magnification and the positive areas in 5 fields/section were measured using ImageJ software (National Institute of Health) [24 (link)]. The area of Ki-67⁺ cells was measured in 10 crypts per each section.

Immunohistochemical staining was performed as previously reported [4 (link), 22 (link)]. The following first Abs were used: 3B3 (Seikagaku Corporation, Tokyo, Japan) for paraffin embedded sections, F4/80 (Serotec, Oxford, UK) and CS-56 (Seikagaku Corporation) for frozen sections [23 (link)]. 3B3 recognizes an epitope created following chondroitinase ABC (ChABC) degradation of chondroitin-6 sulfate. CS-56 recognizes an epitope on some intact chondroitin sulfate glycosaminoglycan chains. The number of F4/80⁺ cells per mm² of alveolar wall was determined using light microscopy, in at least 30 randomly chosen high-power fields [4 (link)]. Immunolabeling for CS-56 and 3B3 were used Histofine kit (Nichirei Bioscience, Tokyo, Japan) according to the manufacturer’s instructions. Tissue sections for 3B3 were pre-incubated in vitro with 0.2 U/mL ChABC (Seikagaku Corporation) in Tris–HCl, pH 8.0, for 1 h at 37 °C.